Comprehensive proteomic analysis of the differential expression of 62 proteins following intracortical microelectrode implantation

Intracortical microelectrodes (IMEs) are devices designed to be implanted into the cerebral cortex for various neuroscience and neuro-engineering applications. A critical feature of IMEs is their ability to detect neural activity from individual neurons. Currently, IMEs are limited by chronic failure, largely considered to be caused by the prolonged neuroinflammatory response to the implanted devices. Over the past few years, the characterization of the neuroinflammatory response has grown in sophistication, with the most recent advances focusing on mRNA expression following IME implantation. While gene expression studies increase our broad understanding of the relationship between IMEs and cortical tissue, advanced proteomic techniques have not been reported. Proteomic evaluation is necessary to describe the diverse changes in protein expression specific to neuroinflammation, neurodegeneration, or tissue and cellular viability, which could lead to the further development of targeted intervention strategies designed to improve IME functionality. In this study, we have characterized the expression of 62 proteins within 180 μm of the IME implant site at 4-, 8-, and 16-weeks post-implantation. We identified potential targets for immunotherapies, as well as key pathways that contribute to neuronal dieback around the IME implant.


Overall protein expression
Our starting point for investigation was to combine all of the proteins of interest investigated in this study into one complete analysis of neuroinflammatory markers.To our knowledge, our dataset represents the most comprehensive assessment of neuroinflammatory protein expression following intracortical microelectrode implantation to date.Here, we first compared implanted mouse groups (4WK, 8WK, 16WK) to naïve control mice to evaluate implanted protein expression levels versus the expression levels of healthy tissue in the same region.A total of forty proteins showed differential expression within at least one temporal comparison (Fig. 1A).Table 1 shows the full list of all significant proteins, their functions, as well as a summary of when the proteins were significantly differentially expressed.There were seventeen total differentially expressed proteins in the 4WK time point.Of these seventeen proteins, ten were differentially expressed only in 4WK mice.Six proteins were upregulated in only the 4WK vs. naïve control comparison: ionized calcium binding adapter molecule 1 (IBA1), Ki-67, major histocompatibility complex class II (MHC II), pan cytokeratin (PanCk), secreted phosphoprotein 1 (SPP1), and transmembrane protein 119 (TMEM119).Four proteins were downregulated in only the 4WK vs. naïve control

Categorical analysis of differential protein expression
Proteins that were identified as statistically significantly differentially expressed were broken down into specific categories to analyze the expression changes in specific cell types or pathways.The categories indicate proteins as being related to astrocytes or microglia, peripheral immunity, neuronal and oligodendrocyte health, and autophagy.Functions of these proteins are listed in Table 1.Many microglial and macrophage proteins are included in both the astrocyte or microglia section and the peripheral immune section because microglia and www.nature.com/scientificreports/macrophages are both myeloid cells that have many overlapping markers 22 , but macrophages are peripheral cells that infiltrate the brain tissue through a damaged or leaky BBB 23,24 .Proteins that are involved in endothelial cell function were grouped with the peripheral immune section as their downregulation could result in BBB damage, leading to infiltration of both blood-derived proteins (FN for example) and blood-derived cells (macrophages for example) into the brain parenchyma.

Indicating proteins for astrocytes or microglia
Astrocytes and microglia have important immune functions in the CNS, being the resident immune cells in the brain 2 .Our analysis revealed that seventeen astrocyte or microglia-associated proteins were found to be significantly differentially expressed in at least one temporal comparison (Fig. 2).Three proteins are astrocytespecific: ALDH1L1, GFAP, and VIM.One is microglia-specific: TMEM119.Six proteins are expressed by both microglia and macrophages: CD11b, CD11c, CD45, CD86, cell surface glycoprotein F4/80 (F4/80), IBA1.Seven proteins are expressed by astrocytes, microglia, and macrophages: CD44, Cathepsin D (CTSD), Ki-67, MSR1, SPP1, MerTK, MHC II.More information regarding the functions of these proteins can be found in Table 1.At the 4WK time point, we saw the highest difference in expression between the implanted group and naïve control mice.Six proteins were differentially expressed in only the 4WK vs. naïve control comparison.Five of these proteins were upregulated: IBA1, Ki-67, MHC II, SPP1, and TMEM119.One protein, CD86, was downregulated.Three proteins were differentially expressed in only the 8WK vs. naïve control comparison, all upregulated: CD44, F4/80, and CTSD.Three proteins were differentially expressed in only the 16WK vs. naïve control comparison: ALDH1L1, MerTK, and MSR1, all of which were downregulated.Five proteins were differentially expressed in all three comparisons: CD11b, CD11c, CD45, GFAP, and VIM.CD11b, CD11c, CD45, and GFAP were upregulated at 4WK, 8WK, and 16WK, while VIM was downregulated at all three time points (Fig. 2, Table 1).We did not find any proteins in this sub-grouping to be differentially expressed in two, but not all three, of the examined time points.

Indicating proteins for the peripheral immune system
Bleeding into the injury site may cause new cell populations such as macrophages, T-cells, or neutrophils to contribute to the inflammatory response 25 .There were 21 significant proteins associated with peripheral immunity.Five proteins were upregulated exclusively at the 4WK time point: IBA1, Ki-67, MHC II, PanCk, and SPP1.Four proteins were downregulated exclusively at the 4WK time point: CD3E, CD86, CTLA4, and Ly6G/Ly6C.One protein, FN, was upregulated at both the 4WK and 8WK time points.Six proteins were upregulated in only the 8WK vs. naïve control comparison: CD31, CD34, CD40L, CD44, CTSD, and F4/80.Two proteins were downregulated in only the 16WK time point: MerTK and MSR1.Three immune proteins were upregulated in all three comparisons: CD11b, CD11c, and CD45 (Fig. 3, Table 1).

Indicating proteins for neurons and oligodendrocytes
Many of the proteins in the designed neural panel play a role in maintaining homeostasis in neurons, including proteins that form the neuronal cytoskeleton, synaptic vesicles, and the myelin that enhances action potential propagation 26,27 .All proteins investigated in this study that benefit neuronal health and functionality were included in this category.Of the 62 proteins investigated in the current study, six significantly differentially expressed proteins could be classified as maintaining homeostasis in neurons or oligodendrocytes: MAP2, MBP, NeuN, NfL, OLIG2, and SYP.At the 4WK time point, MBP displayed significant downregulation in expression levels compared to naïve control.By 8WK, there was significant downregulation of all six neuronal health proteins (MAP2, MBP, NeuN, NfL, OLIG2, and SYP).Four out of the six proteins downregulated in the 8WK timepoint continued to be downregulated into the 16WK time point: MAP2, NfL, MBP, and OLIG2.Two proteins: NeuN and SYP, were downregulated at the 8WK time point but showed no differential expression at either the 4WK or 16WK time points (Fig. 4, Table 1).

Indicating proteins for autophagy
Autophagy is the process in which the cell breaks down damaged and dysfunctional substances in the cytoplasm 28 .Nine proteins associated with autophagy were found to be significantly differentially expressed in at least one temporal comparison: ATG5, ATG12, BAG3, BECN1, P62, PLA2G6, TFEB, ULK1, and VPS35.None of the autophagy-related proteins were significantly differentially expressed in the 4WK time point.Eight out of the nine proteins were downregulated at both the 8WK and 16WK time points: ATG5, BAG3, BECN1, P62, PLA2G6, TFEB, ULK1, and VPS35.The one protein that was downregulated at only the 8WK was ATG12 (Fig. 5, Table 1).

Discussion
IMEs face limitations associated with chronic failure, predominantly attributed to the prolonged neuroinflammatory response 2 .The characterization of the neuroinflammatory response has evolved over decades, embracing advanced transcriptomic techniques [13][14][15][16][29][30][31][32][33][34] . While ene expression studies enhance our broad comprehension of the interplay between IMEs and cortical tissue, there is a notable absence of reported advanced proteomic methods to explain the intricate changes in protein expression specific to neuroinflammation, neurodegeneration, or the viability of tissue and cells.This shortfall has likely hampered the development of targeted intervention strategies to enhance IME functionality.
Figure 6 reviews how the biological response to neural implants is typically quantified.The search terms used in PubMed were: "microelectrode" AND ("biological response" OR "inflammation" OR "tissue response" OR "inflammatory response" OR "foreign body response" OR "failure") AND ("brain" OR "cortical" OR "intracortical").This search output a reasonably sized representation of the literature, but this is not an exhaustive list of all IME papers in the field.All papers from this search published in 2000 or later were included in the review (n = 282).The search terms were intended to target experimental papers that developed or characterized microelectrodes and implanted them into either the brain or live neural cells.Any papers that did not fit these requirements (n = 94) were removed.
Of the remaining 188 papers, 39% (73 papers) did not mention the biological response to the implant.Of the 61% of papers (115 papers) that did mention the biological response, only 77% (88 papers) of that subgroup used any quantitative metric to determine the effect of the implant on the tissue.Of the 88 papers that used quantitative methods to characterize the biological response to the implant, 82% (72 papers) used protein expression assays to quantify the response.The 72 proteomic papers represent a relatively high number of papers using protein expression as an indication of the state of the brain tissue.However, 94% of papers (68 in total) that looked into protein expression used a method that measured intensity, such as immunohistochemistry or two-photon microscopy.Analyzing intensity measurements means that the papers were generally unable to look at large numbers of proteins at once due to a limited number of microscope channels.Methods like immunohistochemistry also do not quantify protein counts, but instead estimate based on fluorescent intensity.The average number of proteins quantified in the subgroup that used intensity measurements was 3.6 proteins.The maximum number of proteins quantified using intensity measurements was 15, while the minimum was 1.We found four papers that measured protein expression using methods other than fluorescent intensity-based metrics.Two papers used an enzyme-linked immunosorbent assay (ELISA) to quantify the expression of 2-9 proteins 35,36 .Two papers used a fluorescent bead immunoassay to quantify the expression of 3-4 proteins 37,38 .The proteins quantified in this manner were all cytokines or chemokines.Our study expands on current methods by using the actual protein counts rather than intensity readings and by quantifying 62 proteins at once.In this investigation, we meticulously profiled the expression of approximately 60 proteins within a 180 μm radius of the IME implantation site at 4, 8, and 16 weeks post-implantation to better understand the sub-chronic and chronic neuroinflammatory response to IME implantation.Overall, of the three post-implantation time points investigated in this study (4WK, 8WK, and 16WK), 4WK demonstrates the strongest changes in innate immune marker expression, while 8WK and 16WK exhibit deficits in local neurons and oligodendrocytes (Figs. 1, 2, 3, 4, 5, Table 1).All ten proteins that are only differentially expressed in the 4WK vs naïve control comparison are associated with microglia, macrophages, or peripheral immune cells.The 8WK time point has the largest downregulation of neuronal health and autophagy proteins (Figs. 1C, 4, 5).By 16 weeks post-implantation (16WK), most of the 8WK effects still linger, but several proteins (ATG12, NeuN, SYP) are no longer downregulated (Figs.1D, 4, 5).This could mean that the tissue is healing, but time points past 16 weeks post-implantation would be needed to confirm to what extent the tissue is able to heal.

Proteins associated with astrocytes or microglia
The formation of the tight glial scar that encapsulates the implant is achieved through the migration, expansion, and proliferation of astrocytes upon activation 39 .Two proteins involved in the cytoskeletal expansion of astrocytes are VIM and GFAP 40 .Here, we found GFAP expression to be heavily upregulated at all time points (4WK, 8WK, 16WK), with a ~ 350 to 450% increase in implanted tissue compared to naïve control (Fig. 1).We expected a similar trend with vimentin (VIM), an intermediate filament that plays a similar role in astrocytic activation 41 .However, we found the exact opposite to be true, as vimentin was downregulated in all three measured time points (4WK, 8WK, 16WK) (Fig. 1, Table 1).Vimentin has another role in the motor cortex: it helps to form tight junctions between endothelial cells in blood vessels of the BBB, maintaining the structure that separates the brain parenchyma from circulating blood 42,43 .Implantation of the IME breaks these blood vessels, and the healing process is incredibly slow due to the persistence of the device in the tissue.The BBB is reported to be permeable up to 16 weeks post-implantation 9 .With vimentin being downregulated at 4WK, 8WK, and 16WK time points, the loss around the implant site likely contributes to the leaky vasculature.It is possible that vimentin's overall downregulation in the blood vessels outweighs the upregulation in activated astrocytes.Aldehyde dehydrogenase 1 family member L1 (ALDH1L1) is an enzyme that regulates astrocyte metabolism, cell division, and cell growth.It was only differentially expressed (downregulated) in the 16WK mice (Fig. 1D, Table 1).The function of ALDH1L1 in the CNS is not entirely known, but its downregulation in the 16WK mice may be an indication of injured or diseased state astrocytes 44 .
In the current study, many proteins associated with microglia, specifically CD11b, CD11c, CD45, IBA1, Ki-67, MHC II, SPP1, and TMEM119, were found to be upregulated in the 4WK time point (Figs.1B, 2, Table 1).Microglia are the main phagocytic cell type in neural tissue 45 , and the upregulation of these proteins validates the presence of microglia at the implant site.CD11b has a known role in cell adhesion during inflammation 45,46 .It is likely upregulated to allow for the adhesion of activated microglia and macrophages to the implanted electrodes and to one another, as they aggregate and form a thin layer on the surface of the electrode 47 .CD11c is expressed in a subset of microglia that are believed to have neuroprotective qualities 48 .Moreover, both CD11c and CD11b are expressed by peripheral immune cells such as dendritic cells, while peripheral macrophages also express CD11b.CD45 is considered to have low expression in microglia compared to macrophages and T-cells, and plays a role in adhesion in myeloid cells 49 .Therefore, upregulation of CD45 could be indicative of T-cells and other peripheral immune cells at the microelectrode interface.IBA1 is involved in the membrane ruffling process and phagocytosis in activated microglia 50 , and has been a common marker for total microglial and macrophage population in cortical tissue in immunohistochemical evaluation of the tissue-electrode interfaces 51,52 .MHC II is a protein involved in antigen presentation that is expressed by microglia, astrocytes, and other immune cells 53,54 .Secreted phosphoprotein 1 (SPP1), also known as osteopontin, is secreted by microglia, macrophages, and T-cells, and is involved in the toll-like receptor signaling pathway.It is pro-inflammatory and activates and recruits more microglia to the implant site 55 .Together, MHC II and SPP1 may implicate the roles of innate and adaptive immune system in response to IME implantations.TMEM119 is a protein expressed on microglia that is mainly abundant in resting cells.Upon activation, TMEM119 concentrations are reduced in microglia 56 .TMEM119 is upregulated at only the 4WK time point, which could indicate that by the 8WK time point the microglia have fully activated and lost the surface TMEM119 abundance.Taken together, the listed microglial and astrocytic proteins could be further investigated as a target to mitigate the inflammatory cascade.Ki-67 is Figure 6.Results from the literature review show of 188 papers that characterized implanted microelectrodes.Only 1 paper quantified protein expression with counts, rather than typical intensity readings.This paper measured the expression of 3 proteins.The search terms used in PubMed were: "microelectrode" AND ("biological response" OR "inflammation" OR "tissue response" OR "inflammatory response" OR "foreign body response" OR "failure") AND ("brain" OR "cortical" OR "intracortical").For a complete list of references, see Supplemental Materials.www.nature.com/scientificreports/ a ubiquitous marker for cell proliferation, and suggests active cell proliferation likely by immune cells at the site of implantation 46,57 .Ki-67 is upregulated in only the 4WK time point compared to naïve control (Fig. 1, Table 1).In the adult motor cortex, neurons no longer proliferate, meaning that the Ki-67 is likely expressed in astrocytes, microglia, and infiltrating peripheral immune cells, not neurons.Ki-67 -/-mice have been shown to reduce tumor growth while also inhibiting major histocompatibility complex expression (see MHC II, Table 1).However, Ki-67 is not essential for proliferation to occur 58 .It is not known how Ki-67 targeting would affect traumatic brain injury or IME implantation, but reducing the efficiency of proliferation in immune cells as well as inhibiting major histocompatibility complexes may be beneficial to neural injuries by reducing the amplification of the inflammatory response in the first 4 weeks following the injury.Other proteins in the proliferation pathway, such as NOX4 or CSF1R, may reduce proliferation more effectively compared to Ki-67 knockout.

Proteins associated with the peripheral immune system
Peripheral immune protein expression can be used to quantify the extent of immune activity at the implant site.These proteins are mainly expressed by T-cells, macrophages, dendritic cells, and neutrophils that circulate through the blood and are recruited into the IME implant site.The BBB is reported to reestablish its integrity at approximately 8 weeks post-implantation, with minor leakage continuing into 16 weeks post-implantation 9 .Peripheral immune cells can be both passively and actively recruited to the site of injury following microelectrode implantation.IBA1, Ki-67, MHC II, and SPP1 are upregulated at only the 4WK time point (Fig. 1, Table 1).All four of these proteins are found on microglia and macrophages, and were discussed in the Astrocyte or Microglia Proteins section.Four proteins are downregulated at the 4WK time point: CD3E, CD86, CTLA4, and Ly6G/ Ly6C.CD86 expressed on innate cells binds and activates CTLA4, and it is believed that this binding dampens T-cell activation by keeping CD86 from binding with CD28 59 .The levels of CTLA4 are low in resting T-cells, and the protein is upregulated by T cells as a self-regulating mechanism of the immune system to prevent run-away inflammation 60 .CTLA4 is downregulated, but CD40L, another protein involved in T-cell activation, is upregulated at the 8WK time point (KEGG:04660) (Fig. 1, Table 1) [61][62][63] .The combined low CTLA4 and high CD40L demonstrates that at 8WK time point there might be higher infiltration of activated T-cells at the implant site.
The mice implanted with IMEs for 4WKs or 8WKs have seven proteins associated with peripheral immune cells upregulated compared to naïve control mice (Figs.1B,C, 3, Table 1).However, by 16WK, only three of these proteins remain upregulated (Figs.1D, 3, Table 1).The three peripheral immunity-associated proteins upregulated at all three time points are CD11b, CD11c, and CD45 (Figs. 1, 3).All three of these proteins are expressed in microglia and macrophages (Table 1).Ly6G/Ly6C is expressed in both myeloid-derived suppressor cells and neutrophils 64,65 .This data indicates the persistence of potential innate immune cells such as macrophages, neutrophils, and dendritic cells until 16 weeks of this study.
The upregulation of peripheral pathways indicates the presence of specific cell types, mainly macrophages and T-cells.On the other hand, downregulation of peripheral pathways indicated that these cells do not migrate into the implant site or are selectively cleared by the 4WK time point.T-cells and other peripheral cells such as dendritic cells and neutrophils are relatively uncharacterized in the context of IME implantation and represent a potential emerging area for immunomodulation of the neuroinflammatory response to intracortical microelectrodes.In fact, immunomodulation of many diseases and injury states through T-cell programming is becoming an emerging area for immunoengineering [66][67][68] .Perhaps similar strategies can be adopted for the neural interface.

Proteins associated with neurons and oligodendrocytes
All six measured proteins associated with maintaining the structure of neurons and oligodendrocytes: MAP2, NfL, SYP, NeuN, MBP, and OLIG2, are downregulated in at least one timepoint (Fig. 2, Table 1).The broad downregulation of markers for neuronal health indicates significant deficits in the health and functionality of both neurons and oligodendrocytes.Two neuronal health proteins: synaptophysin (SYP) and neuronal nuclear protein (NeuN), are downregulated in implanted animals at the 8WK time point and are not significantly differentially expressed at the 4WK or 16WK time points.SYP is a protein that lines the synaptic vesicles 27 .Synaptic vesicles are used to transport neurotransmitters to the synaptic terminals.The release of the neurotransmitters from the synaptic vesicles allows for signal transmission from neuron to neuron 69 .Decreased synaptic protein expression could indicated a decrease in neurons or a decrease in synaptic connections between neurons.NeuN is a protein found in neuronal nuclei involved in mRNA splicing and is the most common marker in the IME histology literature for neuronal health and survival 70 .The fact that SYP and NeuN are both downregulated at 8WK and are not significantly differentially expressed at the 16WK time point could indicate neuronal healing of some capacity between 8 and 16 weeks post-implantation.A similar trend of fluctuations in NeuN density in histological evaluation of the IME-tissue interface was reported by Potter et al. 71 .
MAP2, NfL, MBP, and OLIG2 are all downregulated at both the 8WK and 16WK time points (Figs. 1, 2, Table 1).Neurofilament light (NfL) and microtubule-associated protein 2 (MAP2) are both proteins that make up the neuronal cytoskeleton.Degradation of the neuronal cytoskeleton has been linked to the transition between reversible and irreversible damage to brain tissue 72,73 .NfL is downregulated by ~ 50% (FC = 2 -1 ) in implanted animals by 8WK and ~ 62% (FC = 2 -1.4 ) by 16WK (Fig. 1C, D).The increasing decline in NfL expression could be directly linked to decreases in neuron recording performance with time.However, NfL does not represent a target for immunomodulation approaches to mitigate IME performance.The integrity of the oligodendrocytes, which make up the myelin that allows for efficient transmission of action potentials, are also compromised following IME implantation.Myelin basic protein (MBP) is the second most abundant protein in the myelin cells of the central nervous system 74 , and is considered to be essential for the formation of tight myelin sheaths around an axon 75 .MBP is approximately four times more abundant in naïve control animals compared to implanted mice at Vol:.( 1234567890 www.nature.com/scientificreports/4WK, 8WK, and 16WK time points (Fig. 1B-D).This means that ~ 75% (FC = 2 -2 ) of MBP is lost following IME implantation, which would significantly impact the functionality of the cortical neurons and by extension, the ability of IMES to detect single-unit activity.MBP is the only neuronal health protein that begins downregulation as early as the 4WK time point.Oligodendrocyte transcription factor 2 (OLIG2), is a protein that regulates the transcription for myelin-associated proteins.In traumatic brain injury, OLIG2 is upregulated immediately after injury and remains upregulated for up to 3 months, aiding in the remyelination of the tissue 76,77 .In the case of IME implantation, we see the opposite effect with OLIG2 downregulation at both 8 and 16 weeks post-implantation (Figs. 1, 2, Table 1).The permanent presence of the electrode may be preventing successful remyelination.Overall, the neuronal health proteomic data indicates that the health and functionality of neurons and oligodendrocytes are likely the lowest at approximately the 8WK time point.Degradation of the neuronal cytoskeleton (NfL, MAP2) as well as the oligodendrocytes (MBP, OLIG2) begins at the 8WK time point and continues into the 16WK mice.Some components, including SYP and NeuN, are at least partially regenerated by 16 weeks post-implantation, but other components of the cytoskeleton have endured what may be irreversible damage.These structural components (NfL, MAP2, MBP) are degrading and are not being regenerated by 16 weeks post-implantation.Our more complete dataset further questions the validity of using NeuN as the sole marker of neuronal health which has been common practice in the IME literature for some time, as NeuN is not downregulated at the 16WK time point, yet we still see deficits in other neuronal proteins, and 16WKs is associated with chronic recording failure.

Autophagy proteins
Eight autophagy proteins, including ATG5, BAG3, ULK1, and VPS35, are downregulated in the 8WK and 16WK timepoints compared to naïve control mice (Figs.1C, D, 5, Table 1).The downregulation of the 8 autophagy proteins indicates that autophagy is not occurring at a healthy rate in implanted mice by 8 weeks post-surgery.Autophagy removes harmful substances from the cytoplasm, allowing for the recovery of injured cells 28 .Autophagy in neurons is especially important because neurons in the adult motor cortex do not divide or regenerate, so they need to survive the entire lifetime of the organism 78 .The downregulation of autophagy proteins, along with the fact that neurons around the implant site are still dying up to 16 weeks post-implantation 61 , suggest that by the 8WK time point, the autophagy attempts to save the neurons have failed and neurons are likely resorting to apoptosis or necrosis.Though there are no apoptotic proteins quantified in this experiment, MAP2 is known to undergo proteolysis during apoptosis 79 .We found MAP2 to be downregulated in both 8WK and 16WK mice compared to naïve control mice (Figs.1C,D, 4 , Table 1).MAP2 downregulation could indicate that apoptosis is occurring in local neurons.Neuronal dieback is a major concern for IME researchers as well as patients, and autophagy could be a target for the prevention of neuronal death.One study found that overexpression of ATG5, an autophagosomal protein found to be downregulated in our experiment, leads to nearly 20% longer lifespans in mice 80 .A method that promotes autophagy in implanted animals before or during the 8WK and 16WK time points may prevent neuronal dieback over chronic time points.

Implications for future studies
Proteins within the astrocytic, microglial, and peripheral immune sections represent pathways that ideally would not be activated following IME implantation.The knockouts of several key inflammatory genes, including CD14 and C3, have been investigated with some success in IME applications.Ki-67, or a different protein involved in proliferation, may be worth exploring at early time points prior to 4 weeks post-implantation.Our previous understanding of immune cell proteomic activity following IME implantation comes largely from histology studies 2 .Immunohistochemistry has mapped the timelines for microglia, macrophage, and astrocyte aggregation around the implant site.Ravikumar et al. found that microglia and macrophage populations peak at acute time points (2 weeks post-implantation) and slowly decrease over chronic time points (up to 16 weeks postimplantation).Astrocytic aggregation was shown to be highest at acute timepoints (2 weeks post-implantation), then fluctuate slightly around similar values between 4 and 16 weeks post-implantation 11 .Our analysis confirms microglia and macrophage-related proteins (SPP1, MHC II, IBA1) are upregulated at the 4WK timepoint.By the 8WK time point, SPP1, MHC II, and IBA1 all lose significant differential expression, and F4/80 is a microglial/ macrophage protein that becomes significantly upregulated.Astrocytic activation, quantified through GFAP, is consistently upregulated at 4-, 8-, and 16-weeks post-implantation.Other microglia, macrophage, and astrocyterelated proteins such as CD11b/c and CD45 are upregulated at all measured time points of 4-, 8-, and 16-weeks post-implantation.Our analysis confirms that microglia, macrophages, and astrocytes are present at all time points, but brings context into some molecular changes that are occurring in the protein expression of these cells over time.
Our investigation of neuronal health proteins, specifically downregulation of neuronal cytoskeletal and myelin proteins, may indicate that irreversible damage is being done to neurons surrounding the implant.NeuN, which is typically used as a marker for neuronal health, is restored to healthy levels 50+ μm from the implant site by 16 weeks post-implantation 9 .Our protein expression analysis shows that approximately 62% of NfL is lost in 16WK implanted animals.The 62% of NfL lost within 180 μm is unlikely to be contained within the 50 μm radius where neuronal nuclei are depleted.This calls into question how reliable NeuN is as a marker for neuronal health.It could be that neurons are present around the implant but are not functional.Promoting autophagy, specifically ATG5 or ATG12, could improve neuronal survival or functionality.The observed decline in neuronal and autophagy-related proteins may be a turning point for neuronal health that, if prevented, could improve chronic performance beyond 8 weeks post-implantation.Our recommendation for any drug with the intent to prevent neuronal deficits would be to either target continuously up to 8 weeks post-implantation or to target between the 4WK and 8WK time points, which is when the most damage seems to occur (Fig. 4).www.nature.com/scientificreports/With only three time points investigated over ~ 4 months post-implantation in the current study, it is difficult to accurately represent the complete dynamically changing expression patterns from IME implantation to device failure.Transcriptomic studies at the acute time points (< 2 weeks post-implantation) show extreme and highly dynamic gene expression, with prominent changes between days or hours 13,14 .However, many studies have shown initial promise in mitigating the inflammatory response, only to still result in chronic failure in the ability to recording single unit action potentials.Therefore, the goal of this initial seminal study was to evaluate chronic effects, to identify targets at timepoints more relevant to when biological-mediate microelectrode failures most often occur.Future studies could evaluate changes in the protein expression patterns at acute time points to determine if the protein expression is as dynamic and variable as the gene expression.Due to the quantity of inflammatory genes upregulated in acute responses to IME implantation, proteomics at acute time points would likely show the vast majority of the inflammatory proteins upregulated, potentially making it difficult to narrow down a target.Mice 4 weeks post-implantation show 11 proteins associated with immune cells upregulated (Figs. 2, 3).Gene expression shows a decline in immune gene expression from 2 to 4 weeks 16 , suggesting that more immune associated proteins could be upregulated at 2 weeks post-implantation.Our study highlights only the differential expression of proteins beyond 4 weeks, in an effort to determine the long-term changes that could be targeted for chronic interventions.
Our results show that proteomic analysis at 8 and 16 weeks post-implantation have similar protein expression patterns, and are distinctly different than the 4 weeks post-implantation profile.Proteins in the 8 and 16-weeks post-implantation groups seem to approach some form of a "steady state" value (Fig. 1).Future studies could add more time points to determine (1) at what point between 4 and 8 weeks do proteins expression levels reach "steady state" and (2) how long does the observed "steady state" remain beyond what was observed at 16 weeks post-implantation.Essentially, initially evaluating only 4, 8, and 16 weeks post-implantation left unknowns to be further explored between these initial time points.Future studies could prioritizing 0-3, 5-7, and > 16 weeks post-implantation to expand the understanding of protein expression surrounding the IME.
It is well established within the field that standard, mainly immunohistochemical, evaluation of the neuroinflammatory response does not necessarily indicate recording performance 2 .However, prior studies were limited because they only evaluated a small number of proteins and used semi-quantitative intensity analyses.Literature on IME recording failure shows the general trend of a steady decline of active electrode channels over chronic time points, regardless of implant type 7 .We can see the same general trends in certain groups of proteins, specifically neuronal health and autophagy-related proteins (Figs. 4, 5).However, IME recording quality is highly variable between different animals and experimental conditions 81 , and with no recording data from the mice on which we performed the spatial proteomics, it is impossible to directly compare the two metrics.It is also difficult to link specific proteins to recording performance definitively due to the limited time points evaluated.Future studies could evaluate proteomics alongside functional recordings in hopes of connecting the two phenomena.
One improvement within the protein collection that could be made with this study is the addition of segmentation based on distance from the implant, cortical depth, or cell type.Immunohistochemistry often separates intensity readouts based on distance from the implant 9,82 and the addition of distance segmentation to proteomics would provide insights into the radius of immune cell activation and neuronal damage.Additionally, different depths of the motor cortex have different cell populations, and depth has been shown to impact recording performance and inflammation 10,83 .Separating collection based on cortical depth could illuminate proteomic changes in different regions of the cortex.Finally, cell segmentation could provide additional context to these results, as many of these proteins are expressed by more than one cell type, and the function of that protein could vary based on the cell type.This would be especially helpful in our analysis of immune cells, which share many common markers (Table 1).Using a finer resolution than 180 µm could also lead to a better understanding of discrepancies in the NeuN marker and other neuron viability markers.However, based on the technology used in the current study, segmentation into additional bins significantly adds to the cost of the experiments as each collection is hybridized and analyzed on the instrument separately.Unfortunately, cost limited the depth of segmentation for this initial seminal study.
Future studies could also evaluate how protein expression changes with different treatments or materials aimed at mitigating neuroinflammation.One major mitigating technique is the implantation of probes made of a flexible polymeric material rather than the stiff silicon probes that are currently used in clinical settings 9,85,86 .Joseph et al. showed that flexible probes demonstrate a similar transcriptomic response to non-implanted controls by 18 weeks post-implantation 14 .Further evaluation of the proteins involved in the injury caused by flexible probe implantation could aid in the clinical implementation of flexible probes by giving insights into failure mechanisms and neuroinflammation.

Conclusions
The current study is the first large-scale analysis of proteins surrounding the IME implant site.Previously, several studies have measured mRNA expression following IME implantation and have quantified individual protein expression mainly using fluorescent intensity.This study provides the high-throughput benefits of RNAseq while also quantifying the molecules that are most relevant to the inflammatory process: proteins.The combination of the proteomic expression data with existing transcriptomic studies could allow for both transcription and translation timelines to be mapped for individual genes and proteins.The timeline of protein expression surrounding the implant could be used to create gene therapies to improve chronic IME performance, and our analysis of 4, 8, and 16 weeks post-implantation can provide insight into when therapies would be best administered.
Our dataset is intended to be used as a database for researchers trying to implement specific genetic targets to improve IME performance or reduce brain damage following implantation.Not only can we add new potential targets to the field, such as Ki-67, but researchers looking into specific pathways can reference Fig. 1  www.nature.com/scientificreports/ to determine the timeline of drug delivery, identify potential dual delivery targets, and review the other molecules that will be at play during targeting.

Animal preparation
All mice were housed at Case Western Reserve University, and all procedures were performed in compliance with an IACUC-approved protocol number 2013-0106.Our study employed anesthesia and euthanasia methods consistent with the ARRIVE guidelines commonly accepted norms of veterinary best practice.A total of 16 C57BL/6J mice were included in this experiment (N = 4 per group).Animals underwent identical surgical procedures following established laboratory protocols 13,15,29,84,118 , resulting in four IME implants spanning both the left and right motor and sensory cortices (n = 16 implants per group).During the surgeries, animals were anesthetized with isoflurane.For full surgical details, please see the above references.In brief, four craniotomies were performed, 1.5 mm lateral and 1.0 mm anterior and posterior to the bregma.The probes were inserted 1 mm deep into the cortex, with four implants per animal.Kwik-Sil was placed over the cavity, and dental cement was added to secure the probe.Implanted mice were sacrificed via cardiac perfusion (described below) at 4, 8, or 16 weeks following their implantation surgery (N = 4 mice at each time point).These groups will be referred to as 4WK, 8WK and 16WK respectively.These mice were compared to naïve control mice (N = 4), with no implant or craniotomy.

Microelectrodes
The probes in this study were non-functional Michigan-style silicon probes from the Pancrazio and Cogan Laboratories at the University of Texas at Dallas, which have been used in previous studies 15,84 .The probes were washed three times in 95% ethanol and sterilized with cold ethylene oxide gas, in accordance with previous protocols 119 .Probes were 2 mm long, 15 μm thick, and 123 μm wide at the widest portion of the shank.The decision to use non-functional probes was due to this study's focus on the neuroinflammatory response, with plans to explore the link between the protein expression and recording performance in future experiments.

Perfusion and tissue processing
At predetermined endpoints, mice were anesthetized with ketamine and xylazine.An incision was then made through the diaphragm and ribcage to expose the heart.While the heart was still beating, a butterfly needle attached to a peristaltic pump was inserted into the left ventricle.The pump was turned on, allowing the flow of 1X phosphate-buffered saline (PBS) into the aorta.The right atrium was cut to relieve pressure on the heart and allow the outflow of blood and PBS.Once 30 ml of 1× PBS was pumped, another 30 ml of 30% sucrose solution in PBS was pumped into the animal for cryoprotection of the brain.The brain was extracted, suspended in Optimum Cutting Temperature compound (OCT, Sakura Finetek USA, Item Number: 25608-930), and flash-frozen on dry ice.The brains remained at −80 °C until further processing.The brains were then cryo-sectioned into 5 μm sections through the depth of the mouse cortex (~ 1 mm) and placed on glass microscope slides (Fisher Scientific, Item Number: 12-550-15).Tissue mounted on the slides was stored at −80 °C until used for multiomics experiments; note only proteomics will be reported in this manuscript.

Analysis for spatially-resolved protein expression:
Slides were removed from the −80 °C freezer and fixed for 16 h in 10% neutral-buffered formalin.Following fixation, the slides were washed three times, 10 min each, with 1× Tris-buffered saline with 0.1% Tween 20 detergent (TBST).Next, the slides were transferred to 1× Citrate buffer to undergo antigen retrieval, which consisted of 15 min in the TintoRetriever Pressure Cooker (Bio SB, Item Number: BSB 7008) on high temperature and pressure settings.The slides were removed from the pressure cooker and left to sit in the citrate buffer for 25 min at room temperature.Next, slides were washed three times in in 1× TBST for ten minutes each.To prepare for overnight staining, a hydrophobic barrier was created on the slide surrounding the tissue using an ImmEdge ® Hydrophobic Barrier PAP Pen (Vector Laboratories, Item number: H-4000).Slides were stained with morphology markers for cell types of interest: neuronal nuclear protein (NeuN) for neuronal nuclei and glial fibrillary acidic protein (GFAP) for activated astrocytes.Morphological markers allowed for the selection of regions of interest, which were rings with 180 μm radius from the implant site.Slides were also stained with protein reagents provided by NanoString, which contained antibodies for the proteins quantified in this study attached to a fluorescent sequence unique to each protein.The total staining solution consisted of 1:40 anti-GFAP (Alexa Fluor ® 532 GA-5, Item Number: NBP2-33184AF532), 1:100 anti-NeuN (Alexa Fluor ® 647 EPR12763, Item Number: ab190565), and 1:25 of all six protein panels and modules provided by NanoString (described below).Slides were placed in a humidity chamber for 16 h in a 4 °C refrigerator for incubation.
The proteins quantified in this study were selected based on existing panels sold by NanoString.For every protein in the panel, the reagent provided would include an antibody for that specific protein bound with a UVcleavable link to a fluorescent "barcode." This barcode consists of a fluorescent sequence that is unique to the antibody it is bound to.The panels purchased for this study were the Neural Cell Profiling Core (25 proteins, Item Number: 121300120), which was paired with the Glial Cell Subtyping Module (10 proteins, Item Number: 121300125), and Autophagy Module (10 proteins, Item Number: 121300124).This makes for 45 total proteins in the Neural Panel (Table 2).The Immune Cell Profiling Core (23 proteins, Item Number: 121300106) was paired with the Immune Cell Typing Module (7 proteins, Item Number: 121300118) and the Immune Activation Status Module (8 proteins, Item Number: 121300117), for a total of 38 proteins in the Immune Panel.This collection of different cores and modules make up the 62 total proteins quantified in this study.Table 2 lists all the proteins from each panel.CD68, a protein in the Neural Cell Profiling Panel that is used as a marker for activated microglia, was removed from the study because it was unable to be validated after multiple attempts.
Following overnight incubation, slides were washed three times in 1× TBST for 10 min each.The hydrophobic barrier on the slides was filled with 200 μl of 10% neutral-buffered formalin, and slides were fixed for an additional thirty minutes in the humidity chamber.The slides were washed twice in 1× TBST for five minutes each.

Table 2.
All proteins included in this study.Columns are split into the neural and immune cell profiling panels, which are sets of proteins that were measured and analyzed separately.Negative control proteins are highlighted in orange, and housekeeping proteins are highlighted in blue.All other proteins (highlighted in grey/white) were included in differential expression analysis.

Figure 1 .
Figure 1.Results of the differential expression analysis.(A) Venn diagram of the results, where the values represent the number of significantly differentially expressed proteins within the given time point.This diagram does not differentiate between up-or downregulation.(B-D) Volcano plots of each time point, where each point is a protein in the panel.The red dashed line shows the significance threshold of p adjusted = 0.05.All significantly differentially expressed proteins are labeled.One insignificant protein (LC3B, p adjusted = 0.19) was omitted from plot B due to an extremely high log 2 (FC) count of 11.92.

Figure 2 .Figure 3 .
Figure 2. Results of the differential expression analysis of proteins associated with astrocytes and microglia.(A) Venn diagram of the results, where the values represent the number of significantly differentially expressed proteins within the given time point.This diagram does not differentiate between up-or downregulation (B-D) Volcano plots of each time point, where each point is a protein in the panel.The red dashed line shows the significance threshold of p adjusted = 0.05.All significantly differentially expressed proteins are labeled.(E) A heat map of the results, where red/yellow/green/teal represents upregulation and dark blue/purple represents downregulation compared to naïve controls.

Figure 4 .
Figure 4. Results of the differential expression analysis of proteins associated with neurons and oligodendrocytes.(A) Venn diagram of the results, where the values represent the number of significantly differentially expressed proteins within the given time point.This diagram does not differentiate between up-or downregulation.(B-D) Volcano plots of each time point, where each point is a protein in the panel.The red dashed line shows the significance threshold of p adjusted = 0.05.All significantly differentially expressed proteins are labeled.(E) A heat map of the results, where red/yellow/green represents upregulation and teal/blue/purple represents downregulation compared to naïve controls.

Figure 5 .
Figure 5. Results of the differential expression analysis of proteins associated with autophagy.(A) Venn diagram of the results, where the values represent the number of significantly differentially expressed proteins within the given time point.This diagram does not differentiate between up-or downregulation (B-D) Volcano plots of each time point, where each point is a protein in the panel.The red dashed line shows the significance threshold of p adjusted = 0.05.All significantly differentially expressed proteins are labeled.(E) A heat map of the results, where red/yellow/green represents upregulation and teal/blue/purple represents downregulation compared to naïve controls. https://doi.org/10.1038/s41598-024-68017-2

Table 1 .
All proteins included in this study found to show differential expression (DE) in at least one comparison (4WK, 8WK, or 16WK) compared to naïve control.+ indicates upregulation in the implanted group (4WK, 8WK, 16WK), and − indicates downregulation in the implanted group compared to naïve control mice.White indicates no significant differential expression.